Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: Serum-starved A549 cells were incubated (or not) with 1 µM AG1478 or 5 µM PP1 for 30 min. Then, the cells were treated for 30 min. with 100 ng/ml EGF or 1 U/ml GO, as indicated. EGFR was IPed from cell lysates, resolved by SDS-PAGE and IBed for total receptor, total tyrosine phosphorylation (p-EGFR) and specific Tyr-residue phosphorylation level (Y845, Y1068, Y1086, and Y1173). Protein aliquots of the cell lysates were also directly IBed for total and Y416 phosphorylated (active) c-Src (p-Src).

Article Snippet: PP1, a Src family inhibitor (Enzo Life Sciences), was dissolved in DMSO and added to the treatment medium at a final concentration of 5 µM.

Techniques: Incubation, SDS Page

Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: A549 cells were incubated (or not) with 5 µM PP1 for 45 min. and then treated (or not) for 15 min. with 100 ng/ml EGF or 30 min. 1 U/ml GO. A . EGFR was IPed from total cell lysates with the mAb 528 and IBed for Y416 phosphorylated c-Src (p-Src) and for total EGFR, as indicated. B . c-Src was IPed from total cell lysates and IBed for total c-Src and EGFR, as indicated.

Article Snippet: PP1, a Src family inhibitor (Enzo Life Sciences), was dissolved in DMSO and added to the treatment medium at a final concentration of 5 µM.

Techniques: Incubation

Journal:

Article Title: Alix/AIP1 Antagonizes Epidermal Growth Factor Receptor Downregulation by the Cbl-SETA/CIN85 Complex

doi: 10.1128/MCB.24.20.8981-8993.2004

Figure Lengend Snippet: Alix binding to EGFR and ΔEGFR is independent of receptor activation. Alix was transfected into HEK293 cells together with EGFR (A) or ΔEGFR (B). In panel B endogenous EGFR was removed from ΔEGFR lysates as described for Fig. ​Fig.1.1. After 24 h cells were serum starved and received no further treatment (lane 1), were treated with 100 ng of EGF/ml for 5 min (lane 2), or were treated with 10 μM EGFR inhibitor AG1478 (lane 3) or 20 μM src kinase inhibitor PP2 (lane 4) for 1 h in the absence of EGF stimulation to further reduce background EGFR phosphorylation levels. No treatment had an effect on the amount of Alix associated with EGFR or ΔEGFR, indicating that the interaction between Alix and receptors was constitutive. P-TYR, phosphotyrosine.

Article Snippet: For epidermal growth factor (EGF) induction experiments 5 million cells were transfected and incubated at 37°C for 24 h. Cells were serum deprived for 18 to 20 h and subsequently incubated with 50 or 100 ng of recombinant EGF/ml in serum-free medium for 5 min. Recombinant human EGF, EGFR kinase inhibitor AG1478, and src kinase inhibitor PP2 were purchased from Oncogene Science.

Techniques: Binding Assay, Activation Assay, Transfection

Journal: Respiratory Research

Article Title: Activation of c-Src tyrosine kinase mediated the degradation of occludin in ventilator-induced lung injury

doi: 10.1186/s12931-014-0158-2

Figure Lengend Snippet: Effect of occludin and c-Src expressions on 20% cyclic stretching with PP2 and DMSO. MLE-12 cells were exposed on 20% cyclic stretching for 4 h. A , B and C : Representative Western blotting of occludin and total and phosphorylated c-Src expressions and the density of proteins in non-stretching group was used as a standard to compare relative densities in the other groups. * P > 0.05, compared with the DMSO group. ** P < 0.05, ## P < 0.05, # P < 0.05, compared with the PP2 group. Data are representative of 3 independent experiments. D : At the end of cyclic stretching, cells were fixed, blocked and then incubated with occludin primary antibody, performed overnight at 4°C. Cy3-Goat Anti-Rabbit IgG (Red) was used as the secondary antibody. Nuclei were counterstained with DAPI (Blue).

Article Snippet: For inhibitor studies, when MLE-12 cell monolayers on BioFlex plates had grown 85–95%, the cells were serum-deprived for 2 h prior to experiments and treated with Src inhibitor PP2 100 nM and DMSO 30 μL/mL for 30 min at 37°C and 5% CO 2 for cyclic stretching testing [ ].

Techniques: Western Blot, Incubation

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Oxidative stress-induced activation of Abl and Src kinases rapidly induces P-glycoprotein internalization via phosphorylation of caveolin-1 on tyrosine-14, decreasing cortisol efflux at the blood–brain barrier

doi: 10.1177/0271678X18822801

Figure Lengend Snippet: Both Abl kinase and Src kinase are involved in the internalization of P-gp and Cav1 in H2O2-treated hCMEC/D3 cells. (a) Relationship between P-gp efflux transport activity and Cav1 expression amounts in the plasma membrane fraction of hCMEC/D3 cells. P-gp efflux transport activity was taken from Figure 1(b). (​(b)b) Comparison between P-gp and Cav1 in the plasma membrane fraction of H2O2-treated hCMEC/D3 cells. hCMEC/D3 cells were incubated with 0.25 mM, 0.5 mM, 1 mM, 2.5 mM or 5 mM H2O2 for 20 min at 37℃ in ECF buffer. The plasma membrane fraction was isolated with an IVB plasma membrane isolation kit. The plasma membrane fraction of hCMEC/D3 cells was digested with lysyl endopeptidase and trypsin. The protein expression amounts were determined by LC-MS/MS analysis. P-gp and Cav1 expression amounts in the plasma membrane fraction under the H2O2-treated condition were taken from Figures 2(c) and 5(a), respectively. (c, e, g) Effect of Abl kinase knockdown on the protein expression amounts of P-gp, Cav1 and MRP1 in the plasma membrane fraction of H2O2-treated hCMEC/D3 cells. hCMEC/D3 cells were transfected with negative control siRNA or siRNA directed against Abl kinase. At 72 h post-transfection, hCMEC/D3 cells were incubated with 0.5 mM H2O2 for 20 min, and then the plasma membrane fraction was isolated and used for LC-MS/MS analysis. To compare the effect of H2O2 on the internalization of P-gp, Cav1 and MRP1 in the negative control siRNA-transfected cell and Abl kinase siRNA-transfected cells, the H2O2-dependent decrease was calculated by subtracting the ratio of the protein expression amounts between H2O2-treated cells per control cells (percent) from 100%. MRP1 was also quantified as a H2O2 non-sensitive membrane marker, because no internalization or reduction of its efflux transport activity was observed in H2O2-treated hCMEC/D3 cells (Figures 1(d) and 2(d)). (d, f, h) Effect of PP2, a Src kinase inhibitor, on the protein expression amounts of P-gp, Cav1 and MRP1 in plasma membrane fraction of H2O2-treated hCMEC/D3 cells. hCMEC/D3 cells were pre-incubated with 10 µM PP2 or DMSO (vehicle) for 30 min, and then 0.5 mM H2O2 was treated with hCMEC/D3 cells for 20 min. The protein expression amounts of P-gp, Cav1 and MRP1 in the plasma membrane fraction were determined by LC-MS/MS analysis. Each value represents the mean ± SD of 10-12 SRM/MRM transitions in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005 (Student's t-test). N.S: not significant.

Article Snippet: Imatinib mesylate was purchased from Biovision Inc. (CA, USA), Src kinase inhibitor PP2 from Cosmo Bio Co. Ltd (Tokyo, Japan), bis-ANS (4,4′-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) from Invitrogen (CA, USA), and KRIBB3 from Santa Cruz Biotechnology (CA, USA).

Techniques: Activity Assay, Expressing, Incubation, Isolation, Liquid Chromatography with Mass Spectroscopy, Transfection, Negative Control, Marker

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Oxidative stress-induced activation of Abl and Src kinases rapidly induces P-glycoprotein internalization via phosphorylation of caveolin-1 on tyrosine-14, decreasing cortisol efflux at the blood–brain barrier

doi: 10.1177/0271678X18822801

Figure Lengend Snippet: Effect of inhibitors of candidate molecules potentially involved in the reduction of P-gp efflux transport activity by H2O2. hCMEC/D3 cells were pre-incubated with inhibitors of candidate molecules for the indicated period of time at 37℃, and the P-gp efflux activity was measured by vinblastine uptake assay with or without PSC833, candidate molecule inhibitors and 5 mM H2O2. (a) Imatinib (10 µM), an Abl kinase inhibitor, was present during pre-incubation for 30 min and incubation for 20 min for vinblastine uptake assay in ECF buffer. (b) PP2 (10 µM), a Src kinase inhibitor, was present during pre-incubation for 30 min and incubation for 20 min for vinblastine uptake in ECF buffer. (c) SKF-96365 (20 µM), a Ca2 + influx inhibitor, was present during pre-incubation for 10 min and incubation for 20 min for vinblastine uptake in ECF buffer. (d) Bis-ANS (10 µM), an inhibitor of microtubule interaction with MAP4, was present during pre-incubation for 30 min and incubation for 20 min for vinblastine uptake in ECF buffer. (e) KRIBB3 (10 µM), an inhibitor of protein kinase C-dependent phosphorylation of HSPB1, was present during pre-incubation for 4 h in culture medium and incubation for 20 min for vinblastine uptake in ECF buffer. Each value represents the mean ± SD (n = 3). *p < 0.05 (Student's t-test).

Article Snippet: Imatinib mesylate was purchased from Biovision Inc. (CA, USA), Src kinase inhibitor PP2 from Cosmo Bio Co. Ltd (Tokyo, Japan), bis-ANS (4,4′-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) from Invitrogen (CA, USA), and KRIBB3 from Santa Cruz Biotechnology (CA, USA).

Techniques: Activity Assay, Incubation

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Oxidative stress-induced activation of Abl and Src kinases rapidly induces P-glycoprotein internalization via phosphorylation of caveolin-1 on tyrosine-14, decreasing cortisol efflux at the blood–brain barrier

doi: 10.1177/0271678X18822801

Figure Lengend Snippet: Tyr14 Cav1 phosphorylation by both Abl kinase and Src kinase is correlated with the reduction of P-gp efflux activity by H2O2. (a) The effects of imatinib and PP2, Abl kinase and Src kinase inhibitors, respectively, on the protein expression amount of Cav-1. hCMEC/D3 cells were incubated with 0.5 mM H2O2 for 20 min at 37℃ after pre-incubation with 10 µM imatinib or 10 µM PP2 for 30 min in ECF buffer. The whole-cell lysate of hCMEC/D3 cells was digested with lysyl endopeptidase and trypsin. Cav1 protein expression amounts were determined by LC-MS/MS analysis. (b) The effects of imatinib and PP2 on the Tyr14 phosphorylation amount of Cav1. Tyr14 phosphorylation levels of Cav1 in the whole-cell lysate of hCMEC/D3 cells were determined by LC-MS/MS analysis as described above. (c, d) Effect of Abl kinase knockdown with siRNA on the protein expression amount or Tyr14 phosphorylation amount of Cav1. hCMEC/D3 cells were transfected with negative control siRNA or Abl kinase siRNA. At 72 h post transfection, LC-MS/MS analysis was performed to determine the protein expression amount or Tyr14 phosphorylation amount of Cav1 in the whole-cell lysate of hCMEC/D3 cells. (e) Confirmation of Abl kinase knockdown by Western blotting. Whole-cell lysate of hCMEC/D3 cells (10 µg protein) was loaded on each lane. Na+/K+ ATPase was detected as a loading control. Three independent experiments were performed and representative data are shown. (f) Confirmation of the PP2 effect on Src kinase activity. Tyr419 phosphorylation amounts of Src kinase in the whole-cell lysate of hCMEC/D3 cells were determined by LC-MS/MS analysis. (g) Relationship between P-gp efflux transport activity and Tyr6, Tyr14, both Tyr6 and Tyr14, Tyr25 and Tyr42 phosphorylation amounts of Cav1 in 0.25 mM to 5 mM H2O2-treated hCMEC/D3 cells. P-gp efflux transport activity after H2O2 treatment was taken from Figure 1(b). After 20 min incubation of H2O2 in ECF buffer, the whole-cell lysate of hCMEC/D3 cells was digested with lysyl endopeptidase and trypsin. The underlined amino acids in the Cav1 sequence are potential phosphorylation sites. The YVDSEGHLYTVPIR peptide contains two phosphorylation sites, so Tyr6-phosphorylated, Tyr14-phosphorylated and both Tyr6/Tyr14-phosphorylated peptides were separately quantified in this study. The phosphorylation levels of Tyr25 and Tyr42 were under the detection limit in 0.25 mM to 5 mM H2O2-treated hCMEC/D3 cells. The limit of quantification values for Tyr25 and Tyr42 was 0.0100 fmol/µg protein and 0.0244 fmol/µg protein, respectively. Quantitative value determined by LC-MS/MS represents the mean ± SD of 9-12 SRM/MRM transitions in three independent analyses. *p < 0.05, ***p < 0.005. NC siRNA: negative control siRNA-transfected cells; Abl siRNA: Abl kinase siRNA-transfected cells; U.L.Q: under the limit of quantification; R: Pearson’s correlation coefficient.

Article Snippet: Imatinib mesylate was purchased from Biovision Inc. (CA, USA), Src kinase inhibitor PP2 from Cosmo Bio Co. Ltd (Tokyo, Japan), bis-ANS (4,4′-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) from Invitrogen (CA, USA), and KRIBB3 from Santa Cruz Biotechnology (CA, USA).

Techniques: Activity Assay, Expressing, Incubation, Liquid Chromatography with Mass Spectroscopy, Transfection, Negative Control, Western Blot, Sequencing